Solid forms of n-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide

ABSTRACT

The present invention relates to solid state forms of N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide (Compound 1), pharmaceutical compositions thereof and methods therewith.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. 119(e) to provisionalapplication Ser. No. 61/315,885, filed on Mar. 19, 2010. The entirecontents of which is incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to solid state forms ofN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide,pharmaceutical compositions thereof, and methods therewith.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is a recessive genetic disease that affectsapproximately 30,000 children and adults in the United States andapproximately 30,000 children and adults in Europe. Despite progress inthe treatment of CF, there is no cure.

CF is caused by mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene that encodes an epithelial chlorideion channel responsible for aiding in the regulation of salt and waterabsorption and secretion in various tissues. Small molecule drugs, knownas potentiators that increase the probability of CFTR channel openingrepresent one potential therapeutic strategy to treat CF.

Specifically, CFTR is a cAMP/ATP-mediated anion channel that isexpressed in a variety of cells types, including absorptive andsecretory epithelia cells, where it regulates anion flux across themembrane, as well as the activity of other ion channels and proteins. Inepithelia cells, normal functioning of CFTR is critical for themaintenance of electrolyte transport throughout the body, includingrespiratory and digestive tissue. CFTR is composed of approximately 1480amino acids that encode a protein made up of a tandem repeat oftransmembrane domains, each containing six transmembrane helices and anucleotide binding domain. The two transmembrane domains are linked by alarge, polar, regulatory (R)-domain with multiple phosphorylation sitesthat regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with CF, mutations in CFTR endogenously expressed inrespiratory epithelia leads to reduced apical anion secretion causing animbalance in ion and fluid transport. The resulting decrease in aniontransport contributes to enhanced mucus accumulation in the lung and theaccompanying microbial infections that ultimately cause death in CFpatients. In addition to respiratory disease, CF patients typicallysuffer from gastrointestinal problems and pancreatic insufficiency that,if left untreated, results in death. In addition, the majority of maleswith cystic fibrosis are infertile and fertility is decreased amongfemales with cystic fibrosis. In contrast to the severe effects of twocopies of the CF associated gene, individuals with a single copy of theCF associated gene exhibit increased resistance to cholera and todehydration resulting from diarrhea—perhaps explaining the relativelyhigh frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000 diseasecausing mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/app). The most prevalent mutationis a deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na^(+/)2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and thebasolateral membrane K⁺ channels, that are responsible for the uptake ofchloride into the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺-K⁺-ATPase pumpand Cl ion channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺-K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases.

N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(Compound 1) is a potent and selective CFTR potentiator of wild-type andmutant (including e.g., ΔF508, R117H, and G551D) forms of human CFTR.Compound 1 is useful for treatment of adult patients with cysticfibrosis and at least one G551D-CFTR allele.

Accordingly, there is a need for stable solid forms of modulators ofCFTR activity, such as Compound 1, that can be used to modulate theactivity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

SUMMARY OF THE INVENTION

The present invention relates to solid forms ofN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(hereinafter “Compound 1”) which has the structure below:

The solid forms of Compound 1 and pharmaceutically acceptablecompositions thereof are useful for treating or lessening the severityof a variety of CFTR mediated diseases. Compound 1 is known as bothN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamideandN-(5-hydroxy-2,4-di-tert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.

In one aspect, the invention also provides a method of treating orlessening the severity of a disease in a patient comprisingadministering to said patient one of the compositions as defined herein,and said disease is selected from cystic fibrosis, asthma, smoke inducedCOPD, chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldiseasepseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In certain embodiments, the disease is cystic fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary X-Ray powder diffraction pattern of Form C ofCompound 1.

FIG. 2 is an exemplary DSC trace of Form C.

FIG. 3 is an exemplary TGA trace of Form C.

FIG. 4 is an exemplary Raman spectrum of Form C.

FIG. 5 is an exemplary FTIR spectrum of Form C.

FIG. 6 is Solid State NMR Spectrum of Form C.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Processes described herein can be used to prepare the compositions ofthis invention. The amounts and the features of the components used inthe processes would be as described herein.

As used herein “crystalline solids” refers to Compounds or compositionswhere the structural units are arranged in fixed geometric patterns orlattices, so that crystalline solids have rigid long range order. Thestructural units that constitute the crystal structure can be atoms,molecules, or ions. Crystalline solids show definite melting points.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present Compounds arewithin the scope of the invention. All tautomeric forms of the Compound1 are included herein. For example, Compound 1 may exist as tautomers,both of which are included herein:

Additionally, unless otherwise stated, structures depicted herein arealso meant to include Compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, Compound 1, whereinone or more hydrogen atoms are replaced deuterium or tritium, or one ormore carbon atoms are replaced by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of this invention. Such Compounds are useful, forexample, as analytical tools, probes in biological assays, or Compoundswith improved therapeutic profile.

Solid Form C of Compound 1

XRPD (X-ray Powder Diffraction)

The XRPD patterns were acquired at room temperature in reflection modeusing a Bruker D8 Advance diffractometer equipped with a sealed tubecopper source and a Vantec-1 detector. The X-ray generator was operatingat a voltage of 40 kV and a current of 40 mA. The data were recorded ina θ-θ scanning mode over the range of 3°-40° 2θ with a step size of0.014° and the sample spinning at 15 rpm.

In one aspect, the invention includes crystallineN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(Compound 1) characterized as Form C.

In one embodiment of this aspect, Form C is characterized by a peakhaving a 2-Theta value from about 6.0 to about 6.4 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 7.3 to about 7.7 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 8.1 to about 8.5 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 12.2 to about 12.6 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 14.4 to about 14.8 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 17.7 to about 18.1 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 20.3 to about 20.7 degrees in an XRPDpattern. In a further embodiment, Form C is characterized by a peakhaving a 2-Theta value from about 20.7 to about 21.1 degrees in an XRPDpattern.

In another embodiment, Form C is characterized by a peak having a2-Theta value of about 6.2 degrees in an XRPD pattern. In a furtherembodiment, Form C is characterized by a peak having a 2-Theta value ofabout 7.5 degrees in an XRPD pattern. In a further embodiment, Form C ischaracterized by a peak having a 2-Theta value of about 8.3 degrees inan XRPD pattern. In a further embodiment, Form C is characterized by apeak having a 2-Theta value of about 12.4 degrees in an XRPD pattern. Ina further embodiment, Form C is characterized by a peak having a 2-Thetavalue of about 14.6 degrees in an XRPD pattern. In a further embodiment,Form C is characterized by a peak having a 2-Theta value of about 17.9degrees in an XRPD pattern. In a further embodiment, Form C ischaracterized by a peak having a 2-Theta value of about 20.5 degrees inan XRPD pattern. In a further embodiment, Form C is characterized by apeak having a 2-Theta value of about 20.9 degrees in an XRPD pattern.

In another embodiment, Form C is characterized by one or more peaks inan XRPD pattern selected from about 6.2, about 7.5, about 8.3, about12.4, about 14.6, about 17.9, about 20.5 and about 20.9 degrees asmeasured on a 2-Theta scale.

In still another embodiment, Form C is characterized by all of thefollowing peaks in an XRPD pattern: about 6.2, about 7.5, about 8.3,about 12.4, about 14.6, about 17.9, about 20.5 and about 20.9 degrees asmeasured on a 2-Theta scale.

In another embodiment, Form C can be characterized by the X-Ray powderdiffraction pattern depicted in FIG. 1. Representative peaks as observedin the XRPD pattern are provided in Table Ia below. Each peak describedin Table Ia also has a corresponding peak label (A-H), which are used todescribe some embodiments of the invention.

TABLE Ia Representative XRPD peaks for Form C. Peak # Angle 2-θ (°) PeakLabel 1 6.2 A 2 7.5 B 3 8.3 C 4 12.4 D 5 14.6 E 6 17.9 F 7 20.5 G 8 20.9H

In another embodiment, Form C can be characterized by an X-Ray powderdiffraction pattern having the representative peaks listed in Table Ib.

TABLE Ib Further representative XRPD peaks for Form C. Peak # Angle 2-θ(°) 1 6.2 2 7.5 3 8.3 4 11.0 5 12.4 6 14.6 7 16.3 8 17.1 9 17.9 10 18.111 18.7 12 19.5 13 20.5 14 20.9 15 21.3 16 21.5 17 21.8 18 22.1 19 22.420 22.7

In one aspect, Compound 1 Form C can be characterized by an X-Ray powderdiffraction pattern having one or more of peaks A, B, C, D, E, F, G andH as described in Table Ia.

In one embodiment of this aspect, Form C is characterized by peak A. Inanother embodiment, Form C is characterized by peak B. In anotherembodiment, Form C is characterized by peak B. In another embodiment,Form C is characterized by peak C. In another embodiment, Form C ischaracterized by peak D. In another embodiment, Form C is characterizedby peak E. In another embodiment, Form C is characterized by peak F. Inanother embodiment, Form C is characterized by peak G. In anotherembodiment, Form C is characterized by peak H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A and B; A and C; A and D; A and E; Aand F; A and G; A and H; B and C; B and D; B and E; B and F; B and G; Band H; C and D; C and E; C and F; C and G; C and H; D and E; D and F; Dand G; D and H; E and F; E and G; E and H; F and G; F and H; and G andH.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B and C; A, B and D; A, B and E; A, Band F; A, B and G; A, B and H; A, C and D; A, C and E; A, C and F; A, Cand G; A, C and H; A, D and E; A, D and F; A, D and G; A, D and H; A, Eand F; A, E and G; A, E and H; A, F and G; A, F and H; A, G and H; B, Cand D; B, C and E; B, C and F; B, C and G; B, C and H; B, D and E; B, Dand F; B, D and G; B, D and H; B, E and F; B, E and G; B, E and H; B, Fand G; B, F and H; B, G and H; C, D and E; C, D F; C, D and G; C, D andH; C, E and F; C, E and G; C, E and H; C, F and G; C, F and H; C, G andH; D, E and F; D, E and G; D, E and H; D, F and G; D, F and H; D, G andH; E, F and G; E, F and H, E, G and H; and F, G and H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C and D; A, B, C and E, A, B, Cand F; A, B, C and G; A, B, C and H; A, B, D and E; A, B, D and F; A, B,D and G; A, B, D and H; A, B, E and F; A, B, E and G; A, B, E and H; A,B, F and G; A, B, F and H; A, B, G and H; A, C, D and E; A, C, D and F;A, C, D and G; A, C, D and H; A, C, E and F; A, C, E and G; A, C, E andH; A, C, F and G; A, C, F and H; A, C, G and H; A, D, F and G; A, D, Fand H; A, D, G and H; A, E, F and G; A, E, F and H; A, E, G and H; A, F,G and H; B, C, D and E; B, C, D and F; B, C, D and G; B, C, D and H; B,C, E and F; B, C, E and G; B, C, E and H; B, C, F and G; B, C, F and B,C, G and H; B, D, E and F; B, D, E and G; B, D, E and H; B, D, F and G;B, D, F and H; B, D, G and H; B, E, F and G; B, E, F and H; B, E, G andH; B, F, G and H; C, D, E and F; C, D, E and G; C, D, E and H; C, D, Fand G; C, D, F and H; C, D, G and H; C, E, F and G; C, E, F and H; C, E,G and H; C, F, G and H; D, E, F and G; D, E, F and H; D, E, G and H; D,F, G and H; and E, F, G and H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D and E; A, B, C, D and F; A,B, C, D and G; A, B, C, D and H; A, B, C, E and F; A, B, C, E and G; A,B, C, E and H; A, B, C, F and G; A, B, C, F and H; A, B, C, G and H; A,B, C, E and F; A, B, C, E and G; A, B, C, E and H; A, B, C, F and G; A,B, C, F and H; A, B, C, G and H; A, B, D, E and F; A, B, D, E and G; A,B, D, E and H; A, B, D, F and G; A, B, D, F and H; A, B, D, G and H; A,B, E, F and G; A, B, E, F and H; A, B, E, G and H; A, B, F, G and H; A,C, D, E and F; A, C, D, E and G; A, C, D, E and H; A, C, D, F and G; A,C, D, F and H; A, C, D, G and H; A, C, E, F and G; A, C, E, F and H; A,C, E, G and H; A, C, F, G and H; A, D, E, F and G; A, D, E, F and H; A,D, E, G and H; A, D, F, G and H; A, E, F, G and H; B, C, D, E and F; B,C, D, E and G; B, C, D, E and H; B, C, D, F and G; B, C, D, F and H; B,C, D, G and H; B, C, E, F and G; B, C, E, F and H; B, C, E, G and H; B,C, F, G and H; B, D, E, F and G; B, D, E, F and H; B, D, E, G and H; B,D, F, G and H; B, E, F, G and H; C, D, E, F and G; C, D, E, F and H; C,D, E, G and H; C, D, F, G and H; C, E, F, G and H; and D, E, F, G and H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D, E and F; A, B, C, D, E andG; A, B, C, D, E and H; A, B, C, D, F and G; A, B, C, D, F and H; A, B,C, D, G and H; A, B, C, E, F and G; A, B, C, E, F and H; A, B, C, E, Gand H; A, B, C, F, G and H; A, B, D, E, F and G; A, B, D, E, F and H; A,B, D, E, G and H; A, B, D, F, G and H; A, B, E, F, G and H; A, C, D, E,F and G; A, C, D, E, F and H; A, C, D, E, G and H; A, C, D, F, G and H;A, C, E, F, G and H; A, D, E, F, G and H; B, C, D, E, F and G; B, C, D,E, F and H; B, C, D, E, G and H; B, C, D, F, G and H; B, C, E, F, G andH; B, D, E, F, G and H; and C, D, E, F, G and H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D, E, F and G; A, B, C, D, E, Fand H; A, B, C, D, E, G and H; A, B, C, D, F, G and H; A, B, C, E, F, Gand H; A, B, D, E, F, G and H; A, C, D, E, F, G and H; and B, C, D, E,F, G and H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having all of the following peaks asdescribed in Table Ia: A, B, C, D, E, F, G and H.

In another aspect, Compound 1 Form C can be characterized by an X-Raypowder diffraction pattern having one or more of peaks that range invalue within ±0.2 degrees of one or more of the peaks A, B, C, D, E, F,G and H as described in Table 1. In one embodiment of this aspect, FormC is characterized by a peak within ±0.2 degrees of A. In anotherembodiment, Form C is characterized by a peak within ±0.2 degrees of B.In another embodiment, Form C is characterized by a peak within ±0.2degrees of B. In another embodiment, Form C is characterized by a peakwithin ±0.2 degrees of C. In another embodiment, Form C is characterizedby a peak within ±0.2 degrees of D. In another embodiment, Form C ischaracterized by a peak within ±0.2 degrees of E. In another embodiment,Form C is characterized by a peak within ±0.2 degrees of F. In anotherembodiment, Form C is characterized by a peak within ±0.2 degrees of G.In another embodiment, Form C is characterized by a peak within ±0.2degrees of H.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A and B; A and C; A and D; A and E; Aand F; A and G; A and H; B and C; B and D; B and E; B and F; B and G; Band H; C and D; C and E; C and F; C and G; C and H; D and E; D and F; Dand G; D and H; E and F; E and G; E and H; F and G; F and H; and G andH, wherein each peak in the group is within ±0.2 degrees of thecorresponding value described in Table Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B and C; A, B and D; A, B and E; A, Band F; A, B and G; A, B and H; A, C and D; A, C and E; A, C and F; A, Cand G; A, C and H; A, D and E; A, D and F; A, D and G; A, D and H; A, Eand F; A, E and G; A, E and H; A, F and G; A, F and H; A, G and H, B, Cand D; B, C and E; B, C and F; B, C and G; B, C and H; B, D and E; B, Dand F; B, D and G; B, D and H; B, E and F; B, E and G; B, E and H; B, Fand G; B, F and H; B, G and H; C, D and E; C, D F; C, D and G; C, D andH; C, E and F; C, E and G; C, E and H; C, F and G; C, F and H; C, G andH; D, E and F; D, E and G; D, E and H; D, F and G; D, F and H; D, G andH; E, F and G; E, F and H, E, G and H; and F, G and H, wherein each peakin the group is within ±0.2 degrees of the corresponding value describedin Table Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C and D; A, B, C and E, A, B, Cand F; A, B, C and G; A, B, C and H; A, B, D and E; A, B, D and F; A, B,D and G; A, B, D and A, B, E and F; A, B, E and G; A, B, E and H; A, B,F and G; A, B, F and H; A, B, G and H; A, C, D and E; A, C, D and F; A,C, D and G; A, C, D and H; A, C, E and F; A, C, E and G; A, C, E and H;A, C, F and G; A, C, F and H; A, C, G and H; A, D, F and G; A, D, F andH; A, D, G and H; A, E, F and G; A, E, F and H; A, E, G and H; A, F, Gand H; B, C, D and E; B, C, D and F; B, C, D and G; B, C, D and H; B, C,E and F; B, C, E and G; B, C, E and H; B, C, F and G; B, C, F and H; B,C, G and H; B, D, E and F; B, D, E and G; B, D, E and H; B, D, F and G;B, D, F and H; B, D, G and H; B, E, F and G; B, E, F and H; B, E, G andH; B, F, G and H; C, D, E and F; C, D, E and G; C, D, E and H; C, D, Fand G; C, D, F and H; C, D, G and H, C, E, F and G; C, E, F and H; C, E,G and H, C, F, G and H; D, E, F and G; D, E, F and H; D, E, G and H; D,F, G and H; and E, F, G and H, wherein each peak in the group is within±0.2 degrees of the corresponding value described in Table Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D and E; A, B, C, D and F; A,B, C, D and G; A, B, C, D and H; A, B, C, E and F; A, B, C, E and G; A,B, C, E and H; A, B, C, F and G; A, B, C, F and H; A, B, C, G and H; A,B, C, E and F; A, B, C, E and G; A, B, C, E and H; A, B, C, F and G; A,B, C, F and H; A, B, C, G and H; A, B, D, E and F; A, B, D, E and G; A,B, D, E and H; A, B, D, F and G; A, B, D, F and H; A, B, D, G and H; A,B, E, F and G; A, B, E, F and H; A, B, E, G and H; A, B, F, G and H, A,C, D, E and F; A, C, D, E and G; A, C, D, E and H; A, C, D, F and G; A,C, D, F and H; A, C, D, G and H; A, C, E, F and G; A, C, E, F and H; A,C, E, G and H; A, C, F, G and H; A, D, E, F and G; A, D, E, F and H; A,D, E, G and H; A, D, F, G and H; A, E, F, G and H; B, C, D, E and F; B,C, D, E and G; B, C, D, E and H; B, C, D, F and G; B, C, D, F and H; B,C, D, G and H; B, C, E, F and G; B, C, E, F and H; B, C, E, G and H; B,C, F, G and H; B, D, E, F and G; B, D, E, F and H; B, D, E, G and H; B,D, F, G and H; B, E, F, G and H; C, D, E, F and G; C, D, E, F and H; C,D, E, G and H; C, D, F, G and H; C, E, F, G and H; and D, E, F, G and H,wherein each peak in the group is within ±0.2 degrees of thecorresponding value described in Table Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D, E and F; A, B, C, D, E andG; A, B, C, D, E and H; A, B, C, D, F and G; A, B, C, D, F and H; A, B,C, D, G and H; A, B, C, E, F and G; A, B, C, E, F and H; A, B, C, E, Gand H; A, B, C, F, G and H; A, B, D, E, F and G; A, B, D, E, F and H; A,B, D, E, G and H; A, B, D, F, G and H; A, B, E, F, G and H; A, C, D, E,F and G; A, C, D, E, F and H; A, C, D, E, G and H; A, C, D, F, G and H;A, C, E, F, G and H; A, D, E, F, G and H; B, C, D, E, F and G; B, C, D,E, F and H; B, C, D, E, G and H; B, C, D, F, G and H; B, C, E, F, G andH; B, D, E, F, G and H; and C, D, E, F, G and H, wherein each peak inthe group is within ±0.2 degrees of the corresponding value described inTable Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having one of the following groups ofpeaks as described in Table Ia: A, B, C, D, E, F and G; A, B, C, D, E, Fand H; A, B, C, D, E, G and H, A, B, C, D, F, G and H; A, B, C, E, F, Gand H; A, B, D, E, F, G and H; A, C, D, E, F, G and H; and B, C, D, E,F, G and H, wherein each peak in the group is within ±0.2 degrees of thecorresponding value described in Table Ia.

In another embodiment of this aspect, Form C is characterized by anX-Ray powder diffraction pattern having all of the following peaks asdescribed in Table Ia: A, B, C, D, E, F, G and H, wherein each peak inthe group is within ±0.2 degrees of the corresponding value described inTable Ia.

Rietveld Refinement of Form C (Compound 1) from Powder

High resolution data were collected for a crystalline powder sample ofCompound 1 Form C (Collection performed at the European SynchrotronRadiation Facility, Grenoble, France) at the beamline ID31. The X-raysare produced by three 11-mm-gap ex-vacuum undulators. The beam ismonochromated by a cryogenically cooled double-crystal monochromator (Si111 crystals). Water-cooled slits define the size of the beam incidenton the monochromator, and of the monochromatic beam transmitted to thesample in the range of 0.5-2.5 mm (horizontal) by 0.1-1.5 mm (vertical).The wavelength used for the experiment was 1.29984(3) Å.

The powder diffraction data were processed and indexed using MaterialsStudio (Reflex module). The structure was solved using PowderSolvemodule of Materials Studio. The resulting solution was assessed forstructural viability and subsequently refined using Rietveld refinementprocedure.

The structure was solved and refined in a centrosymmetric space groupP2₁/c using simulated annealing algorithm. The main building block inform C is a dimer composed of two Compound 1 molecules related to eachother by a crystallographic inversion center and connected via a pair ofhydrogen bonds between the hydroxyl and the amide carbonyl group. Thesedimers are then further arranged into infinite chains and columnsthrough hydrogen bonding, π-π stacking and van der Waals interactions.Two adjacent columns are oriented perpendicular to each other, one alongthe crystallographic direction a, the other along b. The columns areconnected with each other through van der Waals interactions.

The 4-oxo-1H-quinoline group is locked in a nearly coplanar conformationwith the amide group via an intramolecular hydrogen bond. Owing to thecentrosymmetric space group, Form C structure contains two Compound 1molecular conformations related to one another by rotation around theC1-N12 bond.

A powder pattern calculated from the crystal structure of form C and anexperimental powder pattern recorded on powder diffractometer using aflat sample in reflectance mode have been compared. The peak positionsare in excellent agreement. Some discrepancies in intensities of somepeaks exist and are due to preferred orientation of crystallites in theflat sample.

The results of refinement, instrument setup, radiation details, latticeparameters of the resulting crystal are listed below.

TABLE IIa Results of refinement: Final R_(wp): 10.24% Final R_(p): 7.27%Final R_(wp) (without 15.98% Final CMACS: 0.09% background):

TABLE IIb Results of further refinement: Final R_(wp): 10.50% FinalR_(p): 7.49% Final R_(wp) (without 16.41% Final CMACS: 0.09%background):

TABLE III Setup 2θ Range 1.00-50.00 Step Size 0.003 (degrees):(degrees): Excluded Regions: —

TABLE IV Radiation Type: X-ray Source: Synchrotron λ₁ (Å): 1.299840Monochromator: Double Anom. Dispersion: No Angle: 50.379 Polarization:0.950

TABLE V Lattice Parameters (Lattice Type: Monoclinic; Space Group: P2₁/cParameter Value Refined? a 12.211 Å Yes b  5.961 Å Yes c 32.662 Å Yes α90.00° No β 119.62°  Yes γ 90.00° No

In one embodiment, the crystal structure of Compound 1 Form C has amonoclinic lattice type. In another embodiment, the crystal structure ofCompound 1 Form C has a P2₁/c space group. In another embodiment, thecrystal structure of Compound 1 Form C has a monoclinic lattice type anda P2₁/c space group.

In one embodiment, the crystal structure of Compound 1 Form C has thefollowing unit cell dimensions:

-   -   a=12.211 Angstroms    -   b=5.961 Angstroms    -   c=32.662 Angstroms    -   α=90.00°    -   β=119.62°    -   γ=90.00°

Compound 1 Form C can be characterized by an endotherm beginning at 292°C., that plateaus slightly and then peaks at 293° C. as measured by DSC(FIG. 2). Further, this endotherm preceeds an 85% weight loss, asmeasured by TGA (FIG. 3), which is attributed to chemical degradation.

Compound 1 Form C can be characterized by a FT-IR pattern as depicted inFIG. 5 and by raman spectroscopy as depicted by FIG. 4.

Compound 1 Form C can be characterize by solid state a NMR pattern asdepicted in FIG. 6.

In one aspect, the invention includes Pharmaceutical compositionsincluding Compound 1 Form C and a pharmaceutically acceptable adjuvantor carrier. In one embodiment, Compound 1 Form C can be formulated in apharmaceutical composition, in some instances, with another therapeuticagent, for example another therapeutic agent for treating cysticfibrosis or a symptom thereof.

Processes for preparing Compound 1 Form C are exemplified herein.

Methods of treating a CFTR mediated disease, such as cystic fibrosis, ina patient include administering to said patient Compound 1 Form C or apharmaceutical composition comprising Compound 1 Form C.

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

In another aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise any ofthe Compounds as described herein, and optionally comprise apharmaceutically acceptable carrier, adjuvant or vehicle. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents.

It will also be appreciated that certain Compounds of present inventioncan exist in free form for treatment, or where appropriate, as apharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need thereof is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a Compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the Compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by CFTR. Incertain embodiments, the present invention provides a method of treatinga condition, disease, or disorder implicated by a deficiency of CFTRactivity, the method comprising administering a composition comprising asolid state form of Compound 1 described herein (e.g., Compound 1 asForm C) to a subject, preferably a mammal, in need thereof.

In yet another aspect, the present invention provides a method oftreating, or lessening the severity of a condition, disease, or disorderimplicated by CFTR mutation. In certain embodiments, the presentinvention provides a method of treating a condition, disease, ordisorder implicated by a deficiency of the CFTR activity, the methodcomprising administering a composition comprising a compositioncomprising a solid state form of Compound 1 described herein (e.g.,Compound 1 as Form C) to a subject, preferably a mammal, in needthereof.

In another aspect, the invention also provides a method of treating orlessening the severity of a disease in a patient comprisingadministering to said patient one of the compositions as defined herein,and said disease is selected from cystic fibrosis, asthma, smoke inducedCOPD, chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldiseasepseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, Osteoporosis, Osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, and PrimaryCiliary Dyskinesia (PCD), a term for inherited disorders of thestructure and/or function of cilia, including PCD with situs inversus(also known as Kartagener syndrome), PCD without situs inversus andciliary aplasia.

In some embodiments, the method includes treating or lessening theseverity of cystic fibrosis in a patient comprising administering tosaid patient one of the compositions as defined herein. In certainembodiments, the patient possesses mutant forms of human CFTR. In otherembodiments, the patient possesses one or more of the followingmutations ΔF508, R117H, and G551D of human CFTR. In one embodiment, themethod includes treating or lessening the severity of cystic fibrosis ina patient possessing the ΔF508 mutation of human CFTR comprisingadministering to said patient one of the compositions as defined herein.In one embodiment, the method includes treating or lessening theseverity of cystic fibrosis in a patient possessing the G551D mutationof human CFTR comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includestreating or lessening the severity of cystic fibrosis in a patientpossessing the ΔF508 mutation of human CFTR on at least one allelecomprising administering to said patient one of the compositions asdefined herein. In one embodiment, the method includes treating orlessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes treating or lessening the severity ofcystic fibrosis in a patient possessing the G551D mutation of human CFTRon at least one allele comprising administering to said patient one ofthe compositions as defined herein. In one embodiment, the methodincludes treating or lessening the severity of cystic fibrosis in apatient possessing the G551D mutation of human CFTR on both allelescomprising administering to said patient one of the compositions asdefined herein.

In some embodiments, the method includes lessening the severity ofcystic fibrosis in a patient comprising administering to said patientone of the compositions as defined herein. In certain embodiments, thepatient possesses mutant forms of human CFTR. In other embodiments, thepatient possesses one or more of the following mutations ΔF508, R117H,and G551D of human CFTR. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR comprising administering to said patientone of the compositions as defined herein. In one embodiment, the methodincludes lessening the severity of cystic fibrosis in a patientpossessing the G551D mutation of human CFTR comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the ΔF508 mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theΔF508 mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein. In oneembodiment, the method includes lessening the severity of cysticfibrosis in a patient possessing the G551D mutation of human CFTR on atleast one allele comprising administering to said patient one of thecompositions as defined herein. In one embodiment, the method includeslessening the severity of cystic fibrosis in a patient possessing theG551D mutation of human CFTR on both alleles comprising administering tosaid patient one of the compositions as defined herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteoporosis in a patient comprisingadministering to said patient a composition comprising a solid stateform of Compound 1 described herein (e.g., Compound 1 as Form C).

In certain embodiments, the method of treating or lessening the severityof Osteoporosis in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of Osteopenia in a patient comprisingadministering to said patient a composition comprising a solid stateform of Compound 1 described herein (e.g., Compound 1 as Form C).

In certain embodiments, the method of treating or lessening the severityof Osteopenia in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of bone healing and/orbone repair in a patient comprising administering to said patient acomposition comprising a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C).

In certain embodiments, the method of bone healing and/or bone repair ina patient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of reducing boneresorption in a patient comprising administering to said patient acomposition comprising a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C).

In certain embodiments, the method of reducing bone resorption in apatient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of increasing bonedeposition in a patient comprising administering to said patient acomposition comprising a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C).

In certain embodiments, the method of increasing bone deposition in apatient comprises administering to said patient a pharmaceuticalcomposition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of COPD in a patient comprising administering tosaid patient a composition comprising a solid state form of Compound 1described herein (e.g., Compound 1 as Form C).

In certain embodiments, the method of treating or lessening the severityof COPD in a patient comprises administering to said patient apharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of smoke induced COPD in a patient comprisingadministering to said patient a composition comprising a solid stateform of Compound 1 described herein (e.g., Compound 1 as Form C).

In certain embodiments, the method of treating or lessening the severityof smoke induced COPD in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

In some aspects, the invention provides a method of treating orlessening the severity of chronic bronchitis in a patient comprisingadministering to said patient a composition comprising a solid stateform of Compound 1 described herein (e.g., Compound 1 as Form C).

In certain embodiments, the method of treating or lessening the severityof chronic bronchitis in a patient comprises administering to saidpatient a pharmaceutical composition as described herein.

According to an alternative embodiment, the present invention provides amethod of treating cystic fibrosis comprising the step of administeringto said mammal an effective amount of a composition comprising acompound of the present invention.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of the diseases,disorders or conditions as recited above.

Another aspect of the present invention provides a method ofadministering a pharmaceutical composition by orally administering to apatient at least once per day the composition comprising a compositioncomprising a solid state form of Compound 1 described herein (e.g.,Compound 1 as Form C). In one embodiment, the method comprisesadministering a pharmaceutical composition comprising a compositioncomprising a solid state form of Compound 1 described herein (e.g.,Compound 1 as Form C) every 24 hours. In another embodiment, the methodcomprises administering a pharmaceutical composition comprising acomposition comprising a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C) every 12 hours. In a further embodiment,the method comprises administering a composition comprising a solidstate form of Compound 1 described herein (e.g., Compound 1 as Form C)three times per day. In still a further embodiment, the method comprisesadministering a composition comprising a solid state form of Compound 1described herein (e.g., Compound 1 as Form C) every 4 hours.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of the diseases, disorders or conditions as recited above.

In certain embodiments, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C) or a pharmaceutically acceptablecomposition thereof is useful for treating or lessening the severity ofcystic fibrosis in patients within certain genotypes exhibiting residualCFTR activity, e.g., class III mutations (impaired regulation orgating), class IV mutations (altered conductance), or class V mutations(reduced synthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II,III, IV, and V cystic fibrosis Tansmembrane Conductance RegulatorDefects and Opportunities of Therapy; Current Opinion in PulmonaryMedicine 6:521-529, 2000). Other patient genotypes that exhibit residualCFTR activity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, a solid state form of Compound 1 described herein(e.g., Compound 1 as Form C) or a pharmaceutically acceptablecomposition thereof is useful for treating or lessening the severity ofcystic fibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic insufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the Compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

It will also be appreciated that a solid state form of Compound 1described herein (e.g., Compound 1 as Form C) or a pharmaceuticallyacceptable composition thereof can be employed in combination therapies,that is Form C or a pharmaceutically acceptable composition thereof canbe administered concurrently with, prior to, or subsequent to, one ormore other desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with another agentused to treat the same disorder), or they may achieve different effects(e.g., control of any adverse effects). As used herein, additionaltherapeutic agents that are normally administered to treat or prevent aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In one embodiment, the additional agent is an antibiotic. Exemplaryantibiotics useful herein include tobramycin, including tobramycininhaled powder (TIP), azithromycin, aztreonam, including the aerosolizedform of aztreonam, amikacin, including liposomal formulations thereof,ciprofloxacin, including formulations thereof suitable foradministration by inhalation, levoflaxacin, including aerosolizedformulations thereof, and combinations of two antibiotics, e.g.,fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan compound 1, i.e., an agent that has the effect of modulating CFTRactivity. Exemplary such agents include ataluren (“PTC1248®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid), or(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid. In another embodiment, the additional agent is(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.

In another embodiment, the additional agent is a nutritional agent.Exemplary such agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

A solid state form of Compound 1 described herein (e.g., Compound 1 asForm C) or a pharmaceutically acceptable composition thereof may also beincorporated into compositions for coating an implantable medicaldevice, such as prostheses, artificial valves, vascular grafts, stentsand catheters. Accordingly, the present invention, in another aspect,includes a composition for coating an implantable device comprising asolid state form of Compound 1 described herein (e.g., Compound 1 asForm C) or a pharmaceutically acceptable composition thereof, and inclasses and subclasses herein, and a carrier suitable for coating saidimplantable device. In still another aspect, the present inventionincludes an implantable device coated with a composition comprising asolid state form of Compound 1 described herein (e.g., Compound 1 asForm C) or a pharmaceutically acceptable composition thereof, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

In one aspect, the invention provides a kit for use in measuring theactivity of CFTR or a fragment thereof in a biological sample in vitroor in vivo, comprising:

-   -   (i) a composition comprising Compound 1 Form C;    -   (ii) instructions for:        -   a) contacting the composition with the biological sample;            and        -   b) measuring activity of said CFTR or a fragment thereof.

In one embodiment of this aspect, the kit includes instructions for:

-   -   a) contacting an additional compound with the biological sample;    -   b) measuring the activity of said CFTR or a fragment thereof in        the presence of said additional compound; and    -   c) comparing the activity of said CFTR or fragment thereof in        the presence of said additional compound with the activity of        the CFTR or fragment thereof in the presence of the composition        comprising Compound 1 Form C.

In a further embodiment, the step of comparing the activity of said CFTRor fragment thereof provides a measure of the density of said CFTR orfragment thereof.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Methods & Materials

Differential Scanning calorimetry (DSC)

The DSC traces of Form C were obtained using TA Instruments DSC Q2000equipped with Universal Analysis 2000 software. An amount (3-8 mg) ofCompound 1 Form C was weighed into an aluminum pan and sealed with apinhole lid. The sample was heated from 25° C. to 325° C. at 10° C./min.The sample exhibited high melting points which is consistent with highlycrystalline material. In one embodiment, the melting range is about293.3 to about 294.7° C. In a further embodiment, the melting range isabout 293.8° C. to about 294.2° C. In another embodiment, the onsettemperature range is about 292.2° C. to about 293.5° C. In a furtherembodiment, the onset temperature range is about 292.7° C. to about293.0° C.

Thermogravimetric Analysis (TGA)

TGA was conducted on a TA Instruments model Q5000. An amount (3-5 mg) ofCompound 1 Form C was placed in a platinum sample pan and heated at 10°C./min from room temperature to 400° C. Data were collected by ThermalAdvantage Q Series™ software and analyzed by Universal Analysis 2000software.

XRPD (X-Ray Powder Diffraction)

As stated previously, the XRPD patterns were acquired at roomtemperature in reflection mode using a Bruker D8 Advance diffractometerequipped with a sealed tube copper source and a Vantec-1 detector. TheX-ray generator was operating at a voltage of 40 kV and a current of 40mA. The data were recorded in a θ-θ scanning mode over the range of3°-40° 2θ with a step size of 0.014° and the sample spinning at 15 rpm.

Raman and FTIR Spectroscopy

Raman spectra for Compound 1, Form C was acquired at room temperatureusing the VERTEX 70 FT-IR spectrometer coupled to a RAMII FT-Ramanmodule. The sample was introduced into a clear vial, placed in thesample compartment and analyzed using the parameters outlined in thetable below.

Raman Parameters

Parameter Setting Beam splitter CaF₂ Laser frequency 9395.0 cm⁻¹ Laserpower 1000 mW Save data from 3501 to 2.94 cm⁻¹ Resolution 4 cm⁻¹ Samplescan time 64 scans

The FTIR spectra for Compound 1, Form C was acquired at room temperatureusing the Bruker VERTEX 70 FT-IR spectrometer using the parametersdescribed in the table below.

FTIR Parameters

Parameter Setting Scan range 4000-650 cm⁻¹ Resolution 4 cm⁻¹ Scanssample 16 Scans background 16 Sampling mode ATR, single reflection ZnSe

TABLE VI FTIR and Raman peak assignments for Compound 1, Form C: vs =very strong s = strong, m = medium, w = weak intensity. FTIR RamanWavenumber Wavenumber Intensity, Intensity, Peak assignments peak widthpeak width N—H str in 3281 m Not observed —C(═O)—NHR trans UnsaturatedC—H str - substituted 3085 m, 3056 m 3071 w, 2991 w aromatic and olefinAliphatic C—H str 2991 m, 2955 m, 2959 w, 2913 w, 2907 m, 2876 m 2878 wAmide C═O str + 1643 s Not observed Conjugated ketone C═O str Olefin C═Cconjugated with Not observed 1615 s C═O Amide II in 1524 vs 1528 s—C(═O)—NHR trans Benzene ring str 1475 s Not observed Amide III in 1285s 1310 vs —C(═O)—NHR trans Aromatic C—H wag  765 vs Not observedAromatic in-plane bend modes Not observed  748 s

SSNMR (Solid State Nuclear Magnetic Resonance Spectroscopy)

Bruker-Biospin 400 MHz wide-bore spectrometer equipped withBruker-Biospin 4 mm HFX probe was used. Samples were packed into 4 mmZrO₂ rotors and spun under Magic Angle Spinning (MAS) condition withspinning speed of 12.0 kHz. The proton relaxation time was firstmeasured using ¹H MAS T₁ saturation recovery relaxation experiment inorder to set up proper recycle delay of the ¹³C cross-polarization (CP)MAS experiment. The CP contact time of carbon CPMAS experiment was setto 2 ms. A CP proton pulse with linear ramp (from 50% to 100%) wasemployed. The Hartmann-Hahn match was optimized on external referencesample (glycine). TPPM15 decoupling sequence was used with the fieldstrength of approximately 100 kHz. Some peaks from a ¹³C SSNMR spectrumof Compound 1 Form C are given in Table VII.

TABLE VII Listing of some of the SSNMR peaks for Form C. Compound 1 FormC Peak # Chemical Shift [ppm] Intensity Peak Label 1 176.5 17.95 A 2165.3 23.73 B 3 152.0 47.53 C 4 145.8 33.97 D 5 139.3 30.47 E 6 135.421.76 F 7 133.3 35.38 G 8 131.8 21.72 H 9 130.2 21.45 I 10 129.4 29.31 J11 127.7 31.54 K 12 126.8 25.44 L 13 124.8 20.47 M 14 117.0 42.4 N 15112.2 61.08 O 16 34.5 33.34 P 17 32.3 14.42 Q 18 29.6 100 R

In some embodiments, the ¹³C SSNMR spectrum of Compound 1 Form C isincludes one or more of the following peaks: 176.5 ppm, 165.3 ppm, 152.0ppm, 145.8 ppm, 139.3 ppm, 135.4 ppm, 133.3 ppm, 131.8 ppm, 130.2 ppm,129.4 ppm, 127.7 ppm, 126.8 ppm, 124.8 ppm, 117.0 ppm, 112.2 ppm, 34.5ppm, 32.3 ppm and 29.6 ppm.

In some embodiments, the ¹³C SSNMR spectrum of Compound 1 Form Cincludes all of the following peaks: 152.0 ppm, 135.4 ppm, 131.8 ppm,130.2 ppm, 124.8 ppm, 117.0 ppm and 34.5 ppm.

In some embodiments, the ¹³C SSNMR spectrum of Compound 1 Form Cincludes all of the following peaks: 152.0 ppm, 135.4 ppm, 131.8 ppm and117.0 ppm.

In some embodiments, the ¹³C SSNMR spectrum of Compound 1 Form Cincludes all of the following peaks: 135.4 ppm and 131.8 ppm.

In some embodiments, the SSNMR of Compound 1 Form C includes a peak atabout 152.0 ppm, about 135.4, about 131.8 ppm, and about 117 ppm.

In one aspect, the invention includes Compound 1 Form C which ischaracterized by a ¹³C SSNMR spectrum having one or more of thefollowing peaks: C, F, H, I, M, N and P, as described by Table VII.

In one embodiment of this aspect, Form C is characterized by one peak ina ¹³C SSNMR spectrum, wherein the peak is selected from C, F, H, I, M, Nand P, as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C and F; C and H; Cand N; F and H; F and N; and H and N, as described by Table VII. In afurther embodiment, the ¹³C SSNMR spectrum includes the peaks I, M and Pas described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F and H; C, Hand N; and F, H and N, as described by Table VII. In a furtherembodiment, the ¹³C SSNMR spectrum includes the peaks I, M and P asdescribed by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having the following group of peaks: C, F, H and N, asdescribed by Table VII. In a further embodiment, the ¹³C SSNMR spectrumincludes the peaks I, M and P as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C and F; C and H, Cand N; C and I; C and M; or C and P, as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from F and H; F and N; Fand I; F and M; or F and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from H and N; H and I; H andM; or H and P as described by Table VII. In another embodiment of thisaspect, Form C is characterized by a ¹³C SSNMR spectrum having a groupof peaks selected from N and I; N and M; or N and P as described byTable VII. In another embodiment of this aspect, Form C is characterizedby a ¹³C SSNMR spectrum having a group of peaks selected from I and M; Iand P or M and P as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F and H; C, Fand N; C, F and I; C, F and M; or C, F and P as described by Table VII.In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, H and N; C, Hand I; C, H and M; or C, H and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from C, N and I; C, N and M;or C, N and P as described by Table VII. In another embodiment of thisaspect, Form C is characterized by a ¹³C SSNMR spectrum having a groupof peaks selected from C, I and M; or C, I and P as described by TableVII. In another embodiment of this aspect, Form C is characterized by a¹³C SSNMR spectrum having a group of peaks selected from C, M and P asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom F, H, and N; F, H and I; F, H and M; or F, H and P as described byTable VII. In another embodiment of this aspect, Form C is characterizedby a ¹³C SSNMR spectrum having a group of peaks selected from F, N andI; F, N and M; or F, N and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from F, I and M; or F, I and Pas described by Table VII. In another embodiment of this aspect, Form Cis characterized by a ¹³C SSNMR spectrum having a group of peaksselected from F, M and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from H, N and I; H, N and M;or H, N and P as described by Table VII. In another embodiment of thisaspect, Form C is characterized by a ¹³C SSNMR spectrum having a groupof peaks selected from H, I and M; or H, I and P as described by TableVII. In another embodiment of this aspect, Form C is characterized by a¹³C SSNMR spectrum having a group of peaks selected from H, M and P asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom N, I and M; or N, I and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from N, M and P as describedby Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom I, M and P as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, and N; C,F H, and I; C, F H, and M; or C, F H, and P as described by Table VII.In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from F, H, N and I; F,H, N and M; or F, H, N and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from H, N, I and M; H, N, Iand P; or H, N, I and C as described by Table VII. In another embodimentof this aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from N, I, M and P; N, I, M and C; or N, I, Mand F as described by Table VII. In another embodiment of this aspect,Form C is characterized by a ¹³C SSNMR spectrum having a group of peaksselected from I, M, P and C; I, M, P and F; I, M, P and H as describedby Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, H, N and I; C,H, N, and M; or C, H, N, and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from C, N, I and M; C, N, Iand P; or C, N, I and F as described by Table VII. In another embodimentof this aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from C, I, M and P; C, I, M and F; or C, I, Mand H as described by Table VII. In another embodiment of this aspect,Form C is characterized by a ¹³C SSNMR spectrum having a group of peaksselected from C, M, P and F; C, M, P and H; or C, M, P and N asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom F, N, I and M; F, N, I and P; or F, N, I and C as described byTable VII. In another embodiment of this aspect, Form C is characterizedby a ¹³C SSNMR spectrum having a group of peaks selected from F, I, Mand P; F, I, M and C; F, I, M and H; or F, I, M and N as described byTable VII. In another embodiment of this aspect, Form C is characterizedby a ¹³C SSNMR spectrum having a group of peaks selected from F, M, Pand C; F, M, P and H; or F, M, P and N as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from H, I, M and P; H,I, M and C; or H, I, M and F as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from N, M, P and C; N, M, Pand F; or N, M, P and H as described by Table VII. In another embodimentof this aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from N, M, C and F; or N, M, C and H asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom N, M, F and P as described by Table VII. In another embodiment ofthis aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from N, M, H and P as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, H, I and P; C,F, I and P; C, F, N and P or F, H, I and P as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, N and I;C, F, H, N and M; or C, F, H, N and P; C, F, H, I and M; C, F, H, I andP; C, F, H, M and P; C, F, N, I and M; C, F, N, I and P; C, F, N, M andP; C, H, N, I and M; C, H, N, I and P; C, H, N, M and P; C, H, I, M andP; F, H, N, I and M; F, H, N, I and P; F, H, N, M and P; F, H, I, M andP; F, N, I, M and P or H, N, I, M and P as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, N and I;C, F, H, N and M; or C, F, H, N and P as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, H, N, I and M;or C, H, N, I and P as described by Table VII. In another embodiment ofthis aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from C, N, I, M and P; or C, N, I, M and F asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom C, I, M, P and F; or C, I, M, P and H as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, M, P, F and H;or C, M, P, F and N as described by Table VII. In another embodiment ofthis aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from C, P, F, H and I; or C, P, F, H and M asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom F, H, N, I and M; or F, H, N, I and P as described by Table VII. Inanother embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from F, N, I, M and P;or F, N, I, M and C as described by Table VII. In another embodiment ofthis aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from F, I, M, C and H; F, I, M, C and N asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom F, M, P, C and H; F, M, P, C and N, N, I and M; or F, H, N, I and Pas described by Table VII. In another embodiment of this aspect, Form Cis characterized by a ¹³C SSNMR spectrum having a group of peaksselected from H, N, I M, and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from H, I M, P and F asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom H, M, P, C and F as described by Table VII. In another embodimentof this aspect, Form C is characterized by a ¹³C SSNMR spectrum having agroup of peaks selected from H, P, C, F and I as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, N, I, andM; or C, F, H, N, I and P as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from F, H, N, I, M and P asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom H, N, I, M, P and C as described by Table VII. In anotherembodiment of this aspect, Form C is characterized by a ¹³C SSNMRspectrum having a group of peaks selected from N, I, M, P, C and F asdescribed by Table VII. In another embodiment of this aspect, Form C ischaracterized by a ¹³C SSNMR spectrum having a group of peaks selectedfrom M, P, C, F, H and N as described by Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, N, I, andM; C, F, H, N, I and P; C, F, H, N, M and P; C, F, H, I, M and P; C, F,N, I, M and P; C, H, N, I, M and P or F, H, N, I, M and P as describedby Table VII.

In another embodiment of this aspect, Form C is characterized by a ¹³CSSNMR spectrum having a group of peaks selected from C, F, H, N, I, Mand P as described by Table VII.

SYNTHETIC EXAMPLES Example 1 Total Synthesis of4-oxo-1,4-dihydroquinoline-3-carboxylic acid (26)

Procedure for the preparation of ethyl4-oxo-1,4-dihydroquinoline-3-carboxylate (25)

Compound 23 (4.77 g, 47.7 mmol) was added dropwise to compound 22 (10 g,46.3 mmol) with subsurface N₂ flow to drive out ethanol below 30° C. for0.5 hours. The solution was then heated to 100-110° C. and stirred for2.5 hours. After cooling the mixture to below 60° C., diphenyl ether wasadded. The resulting solution was added dropwise to diphenyl ether thathad been heated to 228-232° C. for 1.5 hours with subsurface N₂ flow todrive out ethanol. The mixture was stirred at 228-232° C. for another 2hours, cooled to below 100° C. and then heptane was added to precipitatethe product. The resulting slurry was stirred at 30° C. for 0.5 hours.The solids were then filtrated, and the cake was washed with heptane anddried in vacuo to give compound 25 as brown solid. ¹H NMR (DMSO-d₆; 400MHz) δ 12.25 (s), δ 8.49 (d), δ 8.10 (m), δ 7.64 (m), δ 7.55 (m), δ 7.34(m), δ 4.16 (q), δ 1.23 (t).

Procedure for the preparation of 4-oxo-1,4-dihydroquinoline-3-carboxylicacid (26)

Method 1

Compound 25 (1.0 eq) was suspended in a solution of HCl (10.0 eq) andH₂O (11.6 vol). The slurry was heated to 85-90° C., although alternativetemperatures are also suitable for this hydrolysis step. For example,the hydrolysis can alternatively be performed at a temperature of fromabout 75 to about 100° C. In some instances, the hydrolysis is performedat a temperature of from about 80 to about 95° C. In others, thehydrolysis step is performed at a temperature of from about 82 to about93° C. (e.g., from about 82.5 to about 92.5° C. or from about 86 toabout 89° C.). After stirring at 85-90° C. for approximately 6.5 hours,the reaction was sampled for reaction completion. Stirring may beperformed under any of the temperatures suited for the hydrolysis. Thesolution was then cooled to 20-25° C. and filtered. The reactorcake wasrinsed with H₂O (2 vol×2). The cake was then washed with 2 vol H₂O untilthe pH≧3.0. The cake was then dried under vacuum at 60° C. to givecompound 26.

Method 2

Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10% NaOH (aq)(10 mL) and ethanol (100 mL). The solution was heated to reflux for 16hours, cooled to 20-25° C. and then the pH was adjusted to 2-3 with 8%HCl. The mixture was then stirred for 0.5 hours and filtered. The cakewas washed with water (50 mL) and then dried in vacuo to give compound26 as a brown solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 15.33 (s), δ 13.39 (s),δ 8.87 (s), δ 8.26 (m), δ 7.87 (m), δ 7.80 (m), δ 7.56 (m).

Example 2 Total synthesis ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(1)

Procedure for the preparation of 2,4-di-tert-butylphenyl methylcarbonate (30)

Method 1

To a solution of 2,4-di-tert-butyl phenol, 29, (10 g, 48.5 mmol) indiethyl ether (100 mL) and triethylamine (10.1 mL, 72.8 mmol), was addedmethyl chloroformate (7.46 mL, 97 mmol) dropwise at 0° C. The mixturewas then allowed to warm to room temperature and stir for an additional2 hours. An additional 5 mL triethylamine and 3.7 mL methylchloroformate was then added and the reaction stirred overnight. Thereaction was then filtered, the filtrate was cooled to 0° C., and anadditional 5 mL triethylamine and 3.7 mL methyl chloroformate was thenadded and the reaction was allowed to warm to room temperature and thenstir for an addition 1 hours. At this stage, the reaction was almostcomplete and was worked up by filtering, then washing with water (2×),followed by brine. The solution was then concentrated to produce ayellow oil and purified using column chromatography to give compound 30.¹H NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J=2.4 Hz, 1H), 7.29 (dd, J=8.4, 2.4Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s,9H).

Method 2

To a reactor vessel charged with 4-dimethylaminopyridine (DMAP, 3.16 g,25.7 mmol) and 2,4-ditert-butyl phenol (compound 29, 103.5 g, 501.6mmol) was added methylene chloride (415 g, 313 mL) and the solution wasagitated until all solids dissolved. Triethylamine (76 g, 751 mmol) wasthen added and the solution was cooled to 0-5° C. Methyl chloroformate(52 g, 550.3 mmol) was then added dropwise over 2.5-4 hours, whilekeeping the solution temperature between 0-5° C. The reaction mixturewas then slowly heated to 23-28° C. and stirred for 20 hours. Thereaction was then cooled to 10-15° C. and charged with 150 mL water. Themixture was stirred at 15-20° C. for 35-45 minutes and the aqueous layerwas then separated and extracted with 150 mL methylene chloride. Theorganic layers were combined and neutralized with 2.5% HCl (aq) at atemperature of 5-20° C. to give a final pH of 5-6. The organic layer wasthen washed with water and concentrated in vacuo at a temperature below20° C. to 150 mL to give compound 30 in methylene chloride.

Procedure for the preparation of 5-nitro-2,4-di-tert-butylphenyl methylcarbonate (31)

Method 1

To a stirred solution of compound 30 (6.77 g, 25.6 mmol) was added 6 mLof a 1:1 mixture of sulfuric acid and nitric acid at 0° C. dropwise. Themixture was allowed to warm to room temperature and stirred for 1 hour.The product was purified using liquid chromatography (ISCO, 120 g, 0-7%EtOAc/Hexanes, 38 min) producing about an 8:1-10:1 mixture ofregioisomers of compound 31 as a white solid. ¹H NMR (400 MHz, DMSO-d₆)δ 7.63 (s, 1H), 7.56 (s, 1H), 3.87 (s, 3H), 1.36 (s, 9H), 1.32 (s, 9H).HPLC ret. time 3.92 min 10-99% CH₃CN, 5 min run; ESI-MS 310 mz (MH)⁺.

Method 2

To compound 30 (100 g, 378 mmol) was added DCM (540 g, 408 mL). Themixture was stirred until all solids dissolved, and then cooled to −5-0°C. Concentrated sulfuric acid (163 g) was then added dropwise, whilemaintaining the initial temperature of the reaction, and the mixture wasstirred for 4.5 hours. Nitric acid (62 g) was then added dropwise over2-4 hours while maintaining the initial temperature of the reaction, andwas then stirred at this temperature for an additional 4.5 hours. Thereaction mixture was then slowly added to cold water, maintaining atemperature below 5° C. The quenched reaction was then heated to 25° C.and the aqueous layer was removed and extracted with methylene chloride.The combined organic layers were washed with water, dried using Na₂SO₄,and concentrated to 124-155 mL. Hexane (48 g) was added and theresulting mixture was again concentrated to 124-155 mL. More hexane (160g) was subsequently added to the mixture. The mixture was then stirredat 23-27° C. for 15.5 hours, and was then filtered. To the filter cakewas added hexane (115 g), the resulting mixture was heated to reflux andstirred for 2-2.5 hours. The mixture was then cooled to 3-7° C., stirredfor an additional 1-1.5 hours, and filtered to give compound 31 as apale yellow solid.

Procedure for the preparation of 5-amino-2,4-di-tert-butylphenyl methylcarbonate (32)

2,4-Di-tert-butyl-5-nitrophenyl methyl carbonate (1.00 eq) was chargedto a suitable hydrogenation reactor, followed by 5% Pd/C (2.50 wt % drybasis, Johnson-Matthey Type 37). MeOH (15.0 vol) was charged to thereactor, and the system was closed. The system was purged with N₂ (g),and was then pressurized to 2.0 Bar with H₂ (g). The reaction wasperformed at a reaction temperature of 25° C.+/−5° C. When complete, thereaction was filtered, and the reactorcake was washed with MeOH (4.00vol). The resulting filtrate was distilled under vacuum at no more than50° C. to 8.00 vol. Water (2.00 vol) was added at 45° C.+/−5° C. Theresultant slurry was cooled to 0° C.+/−5. The slurry was held at 0°C.+/−5° C. for no less than 1 hour, and filtered. The cake was washedonce with 0° C.+/−5° C. MeOH/H₂O (8:2) (2.00 vol). The cake was driedunder vacuum (−0.90 bar and −0.86 bar) at 35° C.-40° C. to give compound32. ¹H NMR (400 MHz, DMSO-d₆) δ 7.05 (s, 1H), 6.39 (s, 1H), 4.80 (s,2H), 3.82 (s, 3H), 1.33 (s, 9H), 1.23 (s, 9H).

Once the reaction was complete, the resulting mixture was diluted withfrom about 5 to 10 volumes of MeOH (e.g., from about 6 to about 9volumes of MeOH, from about 7 to about 8.5 volumes of MeOH, from about7.5 to about 8 volumes of MeOH, or about 7.7 volumes of MeOH), heated toa temperature of about 35±5° C., filtered, washed, and dried, asdescribed above.

Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(1)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P® 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to25.0° C.+/−2.5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture.The reaction mixture was washed with water (10.0 vol) 2 times. 2-MeTHFwas added to bring the total volume of reaction to 40.0 vol (˜16.5 volcharged). To this solution was added NaOMe/MeOH (1.7 equiv) to performthe methanolysis. The reaction was stirred for no less than 1.0 hour,and checked for completion by HPLC. Once complete, the reaction wasquenched with 1 N HCl (10.0 vol), and washed with 0.1 N HCl (10.0 vol).The organic solution was polish filtered to remove any particulates andplaced in a second reactor. The filtered solution was concentrated at nomore than 35° C. (jacket temperature) and no less than 8.0° C. (internalreaction temperature) under reduced pressure to 20 vol. CH₃CN was addedto 40 vol and the solution concentrated at no more than 35° C. (jackettemperature) and no less than 8.0° C. (internal reaction temperature) to20 vol. The addition of CH₃CN and concentration cycle was repeated 2more times for a total of 3 additions of CH₃CN and 4 concentrations to20 vol. After the final concentration to 20 vol, 16.0 vol of CH₃CN wasadded followed by 4.0 vol of H₂O to make a final concentration of 40 volof 10% H₂O/CH₃CN relative to the starting acid. This slurry was heatedto 78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 0.0° C.+/−5° C. over 5 hours, andfiltered. The cake was washed with 0.0° C.+/−5.0° C. CH₃CN (5 vol) 4times. The resulting solid (compound 1) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Alternative Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(1)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P® 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to20° C.+/−5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture. Thereaction mixture was washed with water (10.0 vol) 2 times and 2-MeTHF(16.5 vol) was charged to the reactor. This solution was charged with30% w/w NaOMe/MeOH (1.7 equiv) to perform the methanolysis. The reactionwas stirred at 25.0° C.+/−5.0° C. for no less than 1.0 hour, and checkedfor completion by HPLC. Once complete, the reaction was quenched with1.2 N HCl/H₂O (10.0 vol), and washed with 0.1 N HCl/H₂O (10.0 vol). Theorganic solution was polish filtered to remove any particulates andplaced in a second reactor.

The filtered solution was concentrated at no more than 35° C. (jackettemperature) and no less than 8.0° C. (internal reaction temperature)under reduced pressure to 20 vol. CH₃CN was added to 40 vol and thesolution concentrated at no more than 35° C. (jacket temperature) and noless than 8.0° C. (internal reaction temperature) to 20 vol. Theaddition of CH₃CN and concentration cycle was repeated 2 more times fora total of 3 additions of CH₃CN and 4 concentrations to 20 vol. Afterthe final concentration to 20 vol, 16.0 vol of CH₃CN was chargedfollowed by 4.0 vol of H₂O to make a final concentration of 40 vol of10% H₂O/CH₃CN relative to the starting acid. This slurry was heated to78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 20 to 25° C. over 5 hours, andfiltered. The cake was washed with CH₃CN (5 vol) heated to 20 to 25° C.4 times. The resulting solid (compound 1) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Example 3 Procedure for the recrystallization ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(1)

Compound 1 (1.0 eq) was charged to a reactor. 2-MeTHF (20.0 vol) wasadded followed by 0.1N HCl (5.0 vol). The biphasic solution was stirredand separated and the top organic phase was washed twice more with 0.1NHCl (5.0 vol). The organic solution was polish filtered to remove anyparticulates and placed in a second reactor. The filtered solution wasconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0° C. (internal reaction temperature) under reduced pressure to10 vol. Isopropyl acetate (IPAc) (10 vol) was added and the solutionconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0° C. (internal reaction temperature) to 10 vol. The addition ofIPAc and concentration was repeated 2 more times for a total of 3additions of IPAc and 4 concentrations to 10 vol. After the finalconcentration, 10 vol of IPAc was charged and the slurry was heated toreflux and maintained at this temperature for 5 hours. The slurry wascooled to 0.0° C.+/−5° C. over 5 hours and filtered. The cake was washedwith IPAc (5 vol) once. The resulting solid was dried in a vacuum ovenat 50.0° C.+/−5.0° C.

Methods of Making Compound 1 Form C

Form C of Compound 1 was prepared by adding an excess of Compound 1,prepared as in example 3, into acetonitrile, stirring at 90° C. for 3days, and cooling to room temperature. The product was harvested byfiltration, and the purity of the product was confirmed using SSNMR.

What is claimed is: 1-38. (canceled)
 39. CrystallineN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(Compound 1) characterized as Form C, wherein the Form C ischaracterized by all of the following peaks in an XRPD pattern: about6.2, about 7.5, about 8.3, about 12.4, about 14.6, about 17.9, about20.5 and about 20.9 degrees as measured on a 2-Theta scale, and whereinthe Form C is isolated and purified.
 40. A pharmaceutical compositioncomprising Form C according to claim 39, and a pharmaceuticallyacceptable adjuvant or carrier.
 41. CrystallineN-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide(Compound 1) characterized as Form C, wherein the Form C ischaracterized by a peak having a 2-Theta value from about 6.0 to about6.4 degrees in an XRPD pattern, a peak having a 2-Theta value from about7.3 to about 7.7 degrees in an XRPD pattern, a peak having a 2-Thetavalue from about 8.1 to about 8.5 degrees in an XRPD pattern, a peakhaving a 2-Theta value from about 12.2 to about 12.6 degrees in an XRPDpattern, a peak having a 2-Theta value from about 14.4 to about 14.8degrees in an XRPD pattern, a peak having a 2-Theta value from about17.7 to about 18.1 degrees in an XRPD pattern, a peak having a 2-Thetavalue from about 20.3 to about 20.7 degrees in an XRPD pattern, and apeak having a 2-Theta value from about 20.7 to about 21.1 degrees in anXRPD pattern; and wherein the Form C is isolated and purified.